However, the sCLU expression at early stage of HCC progression remains to be clarified. In this study, the alteration of sCLU oncogenicity was firstly evaluated in HCC- and their para-cancerous- tissues. The incidence of sCLU expression in HCC was significantly higher than that in their non-tumorous tissues at message RNA (mRNA) or protein level, gradually increasing with tumor-node-metastasis (TNM) staging. Abnormal sCLU expression was associated with the poor differentiation, TNM stage, and considered as an independent prognostic factor for HCC patients. Furthermore, silencing sCLU gene transcription inhibited the colony formation and proliferation of HCC cells, with decreasing phosphorylation level of AKT and GSK-3β in HCCLM3 cells  No significant difference at staging I was found between NT and HCC. However, the sCLU mRNA level was drastically up-regulated from staging II to IV  than that in the NT group  is shown in Figure V Figure . The sCL2χ=4.651, P=0.031) and advanced TNM stage . However, no significant associations were found between sCLU expression and patients’ age, gender, AFP level, portal vein invasion, HBV infection, tumor size, liver cirrhosis, lymph node metastasis, and gross classification. Furthermore, the Kaplan-Meier survival curves with log-rank tests were performed to evaluate the prognostic value of sCLU , along with the TNM stage .The univariate or multivariate Cox Regression analysis identifying factors associated with 5-year survival is listed in Table q=18.19, P<0.001). Consistently, the sCLU protein expression was also obviously repressed in cells transfected with shRNA-1, in contrast to the control or NC-shRNA group  or the NC-shRNA group  and the NC-shRNA group  or the NC-shRNA group  or the NC-shRNA group . In addition, the tumor morphological alterations of audio-videoailable atypia among the different groups were confirmed by hematoxylin-eosin staining (H&E). Moreover, the sCLU staining in the control or NC-shRNA group was stronger than that in the shRNA-1 group by immunohistochemistry  and performed according to the World Medical Association Declaration of Helsinki . WrittenA total of 60 primary HCC- and their self-matched NT tissues were collected from HCC patients who received hepatectomy at the Affiliated Hospital of Nantong University, Nantong, China from Jan 2008 to Jan 2010. The diagnosis of HCC was confirmed histologically in each patient, and no patient underwent chemotherapy or radiotherapy before the surgery. Clinical information was obtained from medical records and included a 5-year post-surgery follow-up period. For each case, patient's gender, age, TNM stage, tumor size, differentiation, HBsAg infection, portal vein invasion, and lymph node metastasis status were recorded. In addition, 40 pairs of fresh HCC tissues and matched nontumorous tissues were obtained from the patients who underwent hepatectomy at the Affiliated Hospital of Nantong University from Sept 2012 to Sept 2014. After surgery, the liver tissues were immediately frozen at −80°C for further RNA isolation. The diagnosis of HCC, cirrhosis, and viral hepatitis was based on the criteria proposed by the Ministry of Health of the People's Republic of China and the Chinese National Viral Hepatitis Meeting, respectively.TMA chip containing 60 cancerous- and matched NT- tissues were constructed by the Outdo Biotech Company . IHC assay was performed by using the Autostainer Universal Staining System  as a previous study . In brieThe IHC staining results of sCLU expression in TMA section were evaluated independently by two experienced pathologists using a semi-quantitative score system. IHC score of each sample was defined as the percentage sum of positive staining level  and the staining intensity . According to the IHC score, the cancerous tissues were divided into two groups: the low sCLU expression with 0∼2 scores and high expression with 3∼6 scores. Given the differences between duplicate tissue scores, the higher score was taken as the final score.The shRNAs targeting different sites of human sCLU gene according to the GenBank ID 001831.3 were designed and synthesized by Biomics Company . And the most effective shRNA silencing sCLU was screened as the previously described . NC-shRN2. Specific plasmids were transfected into cells with GenJetTM DNA in Vitro Transfection Reagent  according to the manufacturer's instructions. Briefly, cells were planted in 6-well plates at 24h prior to transfection. Fresh culture medium was replaced in each well at 1h before transfection. Then, 200 μL of DMEM mixture containing DNA and GenJetTM (1:3) was cautiously added into each well. After incubating for 12h, the medium was replaced by fresh complete medium. Finally, the transfection efficiency was observed by the fluorescence microscope.Human hepatoma HCCLM3, SMMC7721, MHCC97-H, HepG2, and hepatocyte L02 cells were purchased from Chinese Academy of Sciences  and cultured in Dulbecco's modified Eagle's medium  with 10% fetal bovine serum  at 37°C in a humidified incubator containing 5% CO3 cells/well) transfected with NC-shRNA, shRNA-1 or without transfection were seeded in 6-well plates and continuously incubated for 10 days. During the indicated time, the culture medium was replaced every 3 days. Then, cells were washed by phosphate buffer saline (PBS), fixed by methanol for 30 min, stained with Giemsa . The single colony with more than 50 cells was counted under a stereomicroscope. The experiment was conducted in triplicate.Human hepatoma cells , the cells was incubated in medium containing CCK-8 reagent for 4 h. After that, the absorbance at 450nm was measured by using a microplate reader. The assays were performed in triplicate.Cell proliferation assay was detected by the Cell Counting Kit 8  according to the manufacturer's instructions. In brief, human hepatoma cells were plated in 96-well plates at a density of 5×10TM First Strand cDNA Synthesis Kit . qRT-PCR was conducted by SYBR®Premix Ex TaqTMII  with the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and the melting curves were detected for the amplification specificity. The messenger RNA (mRNA) value was calculated based on the Ct values using the 2−ΔΔCt method [ΔCt = Cttarget gene - CtGAPDH]. The primers were as follows: sCLU, F: 3′-ATCACTGTGACGGTCCCTGTA-5′, and R: 3′-TCACTCCTCCCGGTGCTT-5′; GAPDH, F: 3′-CAAGGTCATCCATGACAACTTTG-5′, and R: 3′-GTCCACCACCCTGTTGCTGTAG-5′.Total RNA was isolated from tissues and cells with Trizol reagent  according to the manufacturer's instructions. Equal amounts of RNA were reverse transcribed into cDNA using the RevertAidProtein samples collected by sodium dodecyl sulfate (SDS) cell lysis buffer  were separated by SDS-polyacrylamide gel electro- phoresis for 1.5 h, and then transferred onto polyvinylidene fluoride membranes after 2 h. Following blocking for 2.5 h, the membranes were incubated in the primary antibody diluents overnight. Moreover, after washing with tris-buffered saline with tween (TBST), membranes were incubated in the secondary antibody diluents for 2 h. Finally, the samples were detected by electrochemiluminescence (ECL) kit . Antibodies were diluted as follows: GAPDH, AKT and GSK-3β ; sCLU ; phos-AKT and phos-GSK-3β ; IgG horseradish peroxidase conjugate .7 cells) that were stably transfected with sCLU-shRNA, NC-shRNA or without transfection were suspended in 200μL DMEM and injected subcutaneously into the right scapular area of nude mice. Tumor size was measured using calipers every 4 days. The tumor volume was calculated as follows: the tumor volume (mm3) = 0.5 (length×width2). The animals were sacrificed at the end of 5 weeks after injection. The xenograft tumors were dissected and fixed in 4% paraformaldehyde, embedded in paraffin, deparaffinized in xylene, and dehydrated in a gradient of ethanol solutions. Finally, histopathological examination was performed with the Hematoxylin & Eosin (H&E) staining.All procedures on mice were performed according to the Guidelines for the Experimental Animals and approved by the Animal Care and Use Committee of Nantong University. The 6 weeks old BALB/c nude mice (n= 18) were provided by the Experimental Animal Center of Nantong University, and randomly assigned into three groups . Briefly, hepatoma cells . After centrifuged at 12,000 rpm for 30 min, the lysate supernatants were harvested. The total protein levels were measured by the Bicinchonininc Acid Protein Assay Kit  according to the manufacturer's instructions.450nm of duplicate samples were detected with a microplate reader .Furthermore, protein level of sCLU was detected by the sCLU Enzyme-Linked Immunosorbent Assay (ELISA) Kit . In brief, 100μL lysate of each group was added into indicated wells and incubated at 37°C for 2 h. Then 100μL of the sCLU antibody solution and the avidin-biotin complex solution was added in sequence. Contents of wells were aspirated and washed 5 times. After that, 100μL of the tetramethyl benzidine development solution and 90μL of Stop solution were added to appropriate wells to stop reaction. Finally, the values at A2 test. Kaplan-Meier analysis with a log-rank test was performed to calculate the survival curves. The univariate and multivariate Cox regression analyses were conducted to evaluate the prognostic value. Comparisons among the Control, NC-shRNA, and shRNA-1 groups were performed by Student's Newman-Keuls (SNK) comparison. P<0.05 was considered to be statistically significant.Data were presented as means ± standard deviation (SD), and analyzed by using the SPSS19.0 Software. The relationships between clinical features and sCLU expression were determined by the χ


Prostate Specific Antigen (PSA) is the most commonly used serum marker for prostate cancer (PCa), although it is not specific and sensitive enough to allow the differential diagnosis of the more aggressive tumors. For that, new diagnostic methods are being developed, such as PCA-3, PSA isoforms that have resulted in the 4K score or the Prostate Health Index (PHI), and PSA glycoforms. In the present study, we have compared the PHI with our recently developed PSA glycoform assay, based on the determination of the α2,3-sialic acid percentage of serum PSA , in a cohort of 79 patients, which include 50 PCa of different grades and 29 benign prostate hyperplasia (BPH) patients. The % α2,3-SA could distinguish high-risk PCa patients from the rest of patients better than the PHI (area under the curve (AUC) of 0.971 vs. 0.840), although the PHI correlated better with the Gleason score than the % α2,3-SA. The combination of both markers increased the AUC up to 0.985 resulting in 100% sensitivity and 94.7% specificity to differentiate high-risk PCa from the other low and intermediate-risk PCa and BPH patients. These results suggest that both serum markers complement each other and offer an improved diagnostic tool to identify high-risk PCa, which is an important requirement for guiding treatment decisions. Prostate cancer (PCa) is an important problem in public health and a major disease that affects men’s health worldwide. It was the most commonly diagnosed male neoplasia in western countries and Japan last year. It is expected that around one of each six men will be diagnosed with PCa during his life. In addition, as the number of older people increase, the incidence of the disease will raise dramatically in the coming decades .The serum marker Prostate Specific Antigen (PSA), adopted in the early 1990’s, has been the widely used and preferred assay for prostate diseases, including PCa, with important levels of success, and represents the gold standard marker as an essential tool for urologists . In addiHowever, the PSA test presents some limitations. It is organ specific but not cancer specific . Serum PNew non-invasive biomarkers with greater sensitivity and specificity that are capable of distinguishing aggressive tumors from indolent ones are required . To imprRegarding proPSA forms, these were first identified in the serum of patients with prostate cancer in 1997 . ProPSAstPSA . PHI rectPSA ,15,16,17tPSA . HoweverN-glycans in patients with high-risk PCa proPSA. Then the Prostate Health Index (PHI) score was calculated [PHI = ([−2]proPSA /fPSA) × p = 0.006) and BPH (p < 0.001). The intermediate-risk PCa group showed also a significant increase of PHI compared with low-risk PCa (p = 0.006) and BPH (p = 0.022). No significant differences were found between high-risk PCa patients and intermediate-risk PCa neither between BPH and low-risk PCa patients. The plot of the PHI score is shown against the concentration of total serum PSA of each sample A and in p < 0.001), while there was no correlation for the other individual groups.PHI values correlated with the tPSA levels of the sample in the high-risk PCa group   D.N = 67, 28 BPH, seven low-risk, 21 intermediate-risk and 11 high-risk PCa) was evaluated. This subcohort reduced basically the number of high-risk PCa patients, which had high levels of tPSA. In this subcohort, there was no correlation of PHI values and tPSA levels within the high-risk group. The AUC of PHI in this subcohort for identifying high-risk PCa was 0.81, slightly lower than when analyzing the whole cohort.Since PHI values of the high risk group were dependent on tPSA values, a subcohort of patients with tPSA levels lower than 13 ng/mL , PHI performance for PCa diagnosing (AUC of 0.694) was still higher than tPSA (AUC of 0.382) and %fPSA (AUC of 0.630). When PHI was assayed to discriminate PCa from BPH, the AUC was of 0.735, sensitivity of 84% and specificity 45%, with a cutoff of 55.7. The diagnostic performance of PHI was higher than tPSA (AUC of 0.506) and %fPSA (AUC of 0.632), in agreement with bibliographic studies. In the subcohort of patients with tPSA levels lower than 13 ng/mL  with a binomial response. After the introduction of PHI and α2,3-sialic acid percentage values, the GLM allowed to classify the patients as high-risk PCa with 100% sensitivity and 94.7% specificity. The cutoff for PHI score was 65.4 and for α2,3-sialic acid percentage of PSA was 29.94%. The model calculates the probability of a patient to be diagnosed as high-risk PCa or not (either low and intermediate-risk PCa or BPH). For a probability equal to, or higher than 23.2% (that corresponds to the point with maximum sensitivity and specificity) the patient will be classified as high-risk PCa with a sensitivity of 100% and a specificity of 94.7%. For a probability lower than 23.2% the patient will be classified either as a low- or intermediate-risk PCa, or a BPH. The probability for each patient is calculated with the following function using the patient values of PHI and α2,3-sialic acid percentage of PSA , where βNew generation of tumor markers for PCa diagnosis should be able to discriminate between patients with aggressive tumors and those without cancer or low aggressive tumors. Thus, the skills required for the new generation of markers of PCa are high sensitivity and specificity for aggressive tumors. This way, an unnecessary biopsy in men who do not have an aggressive or asymptomatic PCa could be avoided ,28. EarlIn the next future, PCa diagnosis and prognosis will probably depend on panels of biomarkers that will allow a more accurate prediction of PCa presence, stage and aggressiveness, so they will be key factors in a clinician making decisions. These markers could include serum non-invasive markers, as well as imaging markers, such as multi-parametric prostate magnetic resonance (mpMRI), which has also been proposed as a means to avoid the incidental detection of low-grade cancers ,30,31.PHI is a simple and affordable blood test that could be used as part of a multivariable approach to screening. In this sense, PHI has shown good performance for PCa diagnosis . Our resHowever, the performance of PHI in identifying high-risk PCa from the non-aggressive PCa and BPHs is much higher than for identifying PCa from BPH in both the whole cohort and the subcohort, which can be explained because PHI correlates with the Gleason score, as has also been described previously by other studies . The potential of % α2,3-SA to identify high-risk PCa has been confirmed in this study. The AUC was 0.97 with a cutoff of 30%, as previously described. Interestingly, % α2,3-SA performance was not influenced by the tPSA levels of the samples, and had the same performance in the subcohort of tPSA levels lower than 13 ng/mL.% α2,3-SA test identifies PSA glycoforms containing α2,3-sialic acid, which have been linked to PCa aggressiveness ,10,33. PThese results highlight that the future of prostate cancer diagnosis might rely on the combination of a panel of markers based on PSA forms that can give accurate molecular diagnosis and staging and indicate the likelihood of aggressive behavior.The study population included 79 patients (29 BPH and 50 PCa) from Hospital Universitari Dr. Josep Trueta  between 2006 and 2013. The study was approved by the Hospital Ethics Committee (Refs. 169.06 and 023.10) and all patients provided written informed consent before being enrolled. Patients’ sera were collected and stored at −80 °C. Urology and Pathology units from Hospital Universitari Dr. J. Trueta  performed the diagnosis using Transrectal Ultrasound-guided biopsy and/or adenomectomy/prostatectomy followed by pathological analysis.The 29 BPH patients of the study (age range 44–76 years old) had a medical follow-up for a minimum of 2 years. 24 BPH patients had, at least, two negative biopsies with no evidence of high-grade Prostatic Intraepithelial Neoplasia (PIN). The 5 BPH left were subjected to prostate surgery  and confirmed not to have prostate cancer by the Pathology Unit.The 50 PCa patients of the study (age range 46–84 years old) were graded according to the Tumor-Node-Metastasis (TNM) classification following the general guidelines of the European Association of Urology. PCa patients were treatment naïve when serum samples were collected, except one PCa patient of the high-risk group, who was receiving hormonal therapy. High-risk PCa group comprised 22 patients with Gleason scores ≥8 (4 + 4) and/or with metastasis. The low-risk PCa group included 7 patients with Gleason scores of ≤6 (3 + 3), tPSA levels <10 ng/mL and clinical stage ≤pT2a. The group of intermediate-risk patients was comprised of 21 patients that did not meet the above criteria. They had Gleason scores of 7 (3 + 4 or 4 + 3) and 6 (3 + 3) and also included a patient with focal Gleason 8, tPSA levels <10 ng/mL and clinical stage pT2a considering his 10-year relapse-free survival. The average of tPSA serum levels for BPH patients was 7.59 ng/mL . The average of tPSA for the PCa groups was: 17.83 ng/ml  for high-risk PCa patients, 6.44 ng/mL  for intermediate-risk PCa patients, and 4.56 ng/mL  for low-risk PCa patients. Sambucus nigra (SNA)-agarose lectin . Eluted unbound and bound chromatographic fractions were collected by centrifugation and quantification of free PSA of these fractions was performed using the Roche ELECSYS platform and used to determine the percentages of fPSA in the unbound fraction, corresponding to α2,3-sialic acid PSA, and in the bound fractions, which correspond to α2,6-sialic acid PSA.The determination of % α2,3-sialic acid of PSA was performed using a previously published method . BrieflyPatient sera were analyzed for total PSA (tPSA), free PSA (fPSA), and [−2]proPSA on the Beckman Coulter Access 2 analyzer using WHO-standard-calibration. The Prostate Health Index (PHI) score was then calculated [PHI = ([−2]proPSA/fPSA) × Statistical analyses of both PHI and % α2,3-SA as PCa biomarkers were performed using IBM SPSS Statistics 23 for Windows and graphics were generated with SPSS software and GraphPad Prism 5 .U test. Receiver operating characteristic (ROC) curves were analyzed for tPSA, fPSA, % α2,3-SA, and PHI for distinguishing between high-risk PCa from the group of low-risk PCa, intermediate-risk PCa, and BPH, and also for distinguishing between PCa from BPH.Patients were classified into four groups  and Shapiro-Wilk and Levene’s tests were used to assess the normality and homoscedasticity of variables. Differences of % α2,3-SA and PHI value between groups were analyzed using a Mann–Whitney Bivariate regression (Pearson correlation) was used to analyze the correlation of % α2,3-SA and PHI with either the Gleason score or the tPSA levels. To combine PHI and % α2,3-SA, a logistic regression was performed, in which the response variable corresponded to the probability that the event of interest was a high-risk PCa  or the group comprising low- and intermediate-risk PCa and BPH . An R statistical package was used to develop a generalized lineal model (GLM) with binomial response. The construction and the comparison of the AUC of the ROC curves were performed using the Epi ,35 and pp < 0.05 was considered statistically significant.In all these analyses,


To compare the detection efficacy of 11C-choline positron emission tomography and computed tomography (PET/CT) with whole-body magnetic resonance imaging (MRI) including diffusion-weighted imaging (DWI) in patients with suspected recurrent prostate cancer.Fifty-seven patients  underwent 11C-choline PET/CT and MRI using T1-weighted (T1w), short-tau inversion recovery (STIR), and DWI. Two readers visually rated suspicious lesions on a 5-point scale in 20 different regions. Clinical follow-up and histopathology served as the standard of reference (SOR).p = 0.07). The PSA level (>2 ng/mL vs ≤2 ng/mL) significantly influenced the overall performance of PET/CT (p = 0.003) and MRI (p = 0.03). PET/CT was significantly superior to MRI in detecting LR (p = 0.03) and bone metastasis (p = 0.02). We found no difference with respect to the detection of LN metastasis (p = 0.65).Fifty patients  had at least one positive lesion according to the SOR. Twenty-four patients had local recurrence (LR), 27 had lymph node (LN) involvement, and 22 had bone metastases. The overall detection rates for PET/CT and MRI on a patient basis were 94% and 88%, respectively (11C-choline PET/CT was superior in the detection of local recurrence and bone metastasis on a regional basis. Whole-body MRI including DWI showed similar diagnostic accuracy only for detecting lymph node metastases. Compared with 11C-choline PET/CT, therefore, whole-body MRI including DWI cannot serve as alternative imaging modality for restaging prostate cancer. Prostate cancer is the third most common cause of death from cancer in men (9.5%) in Europe and is associated with a high rate of recurrence . After pPositron emission tomography (PET) using 11C-choline has been shown to be promising in both staging and follow-up of patients with prostate cancer , 5. PET Whole-body magnetic resonance imaging (MRI) became possible with the development of parallel imaging and high-gradient amplitudes , 10. HigThe aim of our study, therefore, was to compare the detection rate of 11C-choline PET/CT with that of whole-body MRI including DWI in patients with suspected recurrent prostate cancer.In total, 1026 regions  were evaluated in 57 patients. Fifty patients had at least one positive lesion according to the SOR, 153 regions were finally regarded as positive, and 873 as negative. Twenty-four of the 57 patients included in the study showed evidence of local recurrence. Of 456 lymph node regions evaluated in the study, 59 regions in 27 patients were regarded as positive, 397 regions in 30 patients as negative. Seventy of the 456 bone regions in 22 patients were regarded as positive and 386 regions in 35 patients as negative. Seventeen of 57 patients had evidence of extra-pelvic disease. Five patients had metastases in other regions . These lesions were excluded from analysis.11C-choline PET/CT showed positive results in 94.0% (47/50 patients). MRI analysis resulted in a detection rate of 88.0% (44/50 patients). The PAC of both methods was significantly higher in the subgroup of patients with PSA values greater than 2 ng/mL compared with the subgroup with values lower than or equal to 2 ng/mL  was located in the cervical, thoracic or lumbar spine .Regional analysis of all patients also revealed a significantly higher sensitivity of 11C-choline PET/CT compared to MRI for the detection of LR and bone metastases. However, no significant difference was found with respect to LN metastasis. Cohen’s kappa analysis showed only slight agreement between PET/CT and MRI for the detection of LR , fair agreement for LN metastases , and moderate agreement for bone metastasis . Both 11C-choline PET/CT and MRI generally showed a random distribution of false positives and negatives over the LN and bone regions evaluated. However, none of the false negative bone regions on MRI  probably owing to the high PSA level in our study cohort . HoweverLesions to the lungs and visceral organs were relatively infrequent in our study. However, we excluded those from analysis because of inherent limitations of both MRI (concerning the lung lesions) and PET (concerning liver lesions). Compared to PET/CT, the MRI acquisition time was significantly longer which represents a potential limitation regarding patient throughput. Ten patients had to be excluded from the study because of general MRI contraindications also representing a limitation of this imaging method.However, the diagnosis of LR after radical prostatectomy is often challenging. Souvatzoglou et al. found that only seven (19%) of 37 patients with low PSA values  who were referred for salvage radiotherapy showed an increased uptake in the prostatectomy bed using 11C-choline PET/CT . PanebiaA wide variety of entities can mimic recurrent prostate cancer on MRI. Retained seminal vesicles, fibrotic scaring, a prominent peri-prostatic venous plexus, and granulation tissue can all lead to false positive results . In addiThe evaluation of LN metastases is crucial in restaging patients with PSA failure after radical prostatectomy. Recent studies have evaluated the diagnostic performance of MRI with DWI and 11C-choline PET/CT in preoperative lymph node staging, yielding varying results. In a patient-based analysis of 132 patients, Beheshti et al. obtained a sensitivity of 66% and specificity of 96% in lymph nodes larger than 5 mm when using 11C-choline PET/CT . In a coMRI with DWI is increasingly being used to assess bone marrow and metastatic disease, because it is sensitive to bone marrow cell density, the relative proportions of fat and marrow cells, water content, and bone marrow perfusion . In a re. All false negative bone lesions on MRI were located in sites outside of the cervical, thoracic and lumbar spine, with a lower marrow content in relation to cortical structures.As a general rule in DWI, however, lytic bony metastases are seen more clearly than pure sclerotic metastases, because of the lower water and cellular content of sclerotic and treated lesions . As a reThis study has certain limitations. The high mean PSA values indicate a clear selection bias for which a high positive rate was to be expected. Most patients were at high risk of recurrent disease, with a high prevalence of metastases at the time of recurrence. Most of the patients had at least one positive lesion. Thus, the “true” specificity of the methods cannot be determined. A histological examination in all patients would have been optimal but was not possible for practical and ethical reasons.Instead of a lesion-based analysis of LN metastases, we made region- and patient-based comparisons between imaging and SOR. We chose this approach in consideration of the fact that it is technically extremely challenging to map multiple LNs exactly within a field and correlate each LN with imaging and SOR. However, even this field-wise approach is prone to error when comparing imaging results with follow-up studies.The use of dedicated sequences for the pelvis, such as DCE imaging, could have improved the accuracy of LR detection. However, we did not use such techniques, in order to stay within a reasonable time frame for whole-body imaging. Significantly more time was needed for MR image acquisition compared to that of PET/CT.11C-choline PET/CT was superior to whole-body MRI with DWI in the detection of local recurrence and bone metastasis on a regional basis. The two methods showed equal diagnostic accuracy for detecting lymph node metastases. In summary, whole-body MRI with DWI cannot be recommended as an alternative to 11C-choline PET/CT. In initial studies, 68Ga-PSMA PET/CT was shown to further enhance the accuracy of PET/CT for recurrent prostate cancer.Fifty-seven patients underwent 11C-Choline PET/CT for restaging of PC, which was sort of “routine” in the setting of suspected recurrence of PC during the time of data acquisition. MRI was added as part of our study protocol. Recruitment was performed in an outpatient clinic based on willingness to participate and on the absence of MRI contraindications. During the enrolment period, 10 patients were not eligible for the study because of MRI contraindications . Only patients who had undergone radical prostatectomy were eligible for the study. Patients after chemotherapy or after second line hormonal treatment were excluded. No further patient selection was performed to avoid bias and to ensure a broad spectrum of PSA-values. All patients had MRI and 11C-choline PET/CT scans within 1-3 days (mean 1.1 day). For every patient the imaging time needed for 11C-Choline PET/CT and MRI was analyzed . The mean PSA was 29.9 ng/mL (range 1.0-670 ng/mL). The institutional review board approved the study and all patients gave their informed consent. Table 2 were acquired. The selection of a high b-value of 500 s/mm2 was based on a trade-off between signal intensity and adequate diffusion strength and was consistent with recommendations given in a consensus paper on the use of DWI in cancer patients [MRI was performed using a 1.5T MR scanner . Two anterior 6-element body matrix coils were used in conjunction with two posterior spine clusters (three channels each) to optimize the signal-to-noise ratio (SNR). In addition, a 12-element head matrix coil, a 4-element neck matrix coil, and a 6-element peripheral matrix coil were used. The maximum imaging range in the z-axis was 160 cm with automated table motion. A total of four axial stacks encompassing the base of the skull to the proximal thighs were acquired. According to the convention derived from various reports in the literature , 14, 35,patients .The other technical parameters were as follows: 5 mm slice thickness without intersection gap; number of excitations 3; and generalized autocalibrating partially parallel acquisition (GRAPPA). DWI was achieved by applying pairs of motion-probing gradients before and after the 180° radiofrequency pulse of the spin-echo T2-weighted sequence in three orthogonal directions. ADC maps were calculated from all diffusion weightings and directions on a voxel-by-voxel basis. Conventional T1-weighted (T1w) turbo spin echo (TSE) and short-tau inversion recovery (STIR) images were acquired from the base of the skull to the proximal thigh in axial orientation and for the whole spine in the sagittal plane.2 was converted to 11C-CH3I by the catalytic gas-phase iodination reaction via 11C-CH4 (GE MeI MicroLab). 11C-CH3I, swept with a helium flow at 50 ml/min, was passed through a Light-CM cartridge loaded with N,N-dimethylethanolamine (25 μl). The column was washed with 10 ml ethanol followed by 10 ml water before eluting the product with isotonic saline (2-5 ml of 0.9% NaCl) through a Millipore filter  into a sterile vial. Quality control was performed using HPLC .With minor modifications, 11C-choline was produced according to the method described by Hara et al. . In briePatients fasted for at least 6 hours before 11C-choline PET. Five minutes after the injection of 600-900 MBq 11C-choline, patients underwent PET/CT (neck to pelvis) in a Siemens Sensation 16 Biograph PET/CT scanner. A diagnostic CT scan was performed prior to PET in the portal venous phase (80 s) after the intravenous injection of contrast agent (Imeron 300) . All patients received diluted oral contrast  and rectal filling (100-150 ml). PET scans were acquired in 3D-mode with an acquisition time of 3 minutes per bed position. Iterative reconstruction of the emission data was performed using an ordered-subsets expectation maximization (OSEM) algorithm.MRI and 11C-choline PET/CT scans were analyzed independently by two readers (JCS and HW). The readers were blinded to patient data except for the history of prostate cancer with suspected recurrence on basis of PSA elevation. To avoid any learning bias, all datasets were analyzed in random order with an interval of eight weeks between PET and MRI data.The presence or absence of local recurrence (LR), lymph node (LN), bone or other metastases was evaluated for each patient and imaging modality. As it was not feasible to compare imaging modalities with the standard of reference (SOR) for single LN or bone metastases, we evaluated specific regions as a whole . Any other visceral metastases (e.g. in the lungs or liver) were evaluated as one additional region per patient. For each of the anatomical locations, the probability of local recurrence and/or metastases was evaluated using a qualitative 5-point scoring system: definitely present (score 5); probably present (score 4); equivocal (score 3); probably absent (score 2); and definitely absent (score 1). Discordant readings from the two observers were resolved by consensus for each modality.Focal 11C-choline activity in the prostatectomy bed greater than adjacent background uptake and not due to radiotracer excreted in the urine was rated 4 or 5, regardless of any corresponding structural abnormality. LNs were rated 4 or 5 if distinct focal activity greater than adjacent background uptake on PET images corresponded to a visible LN on CT, regardless of size; LNs distal to the mid external iliac chains were excluded because increased radiotracer activity in these regions is common . Focal sMass-like low T2 signal intensity with diffusion restriction in the prostatectomy bed or in the area of the former neurovascular bundle was taken to be significant for LR and was subsequently rated as 4 or 5. LN involvement was rated on the basis of at least one lymph node per region showing increased signal intensity on inverted b-images: short-axis diameter > 10 mm (score 5), 8-9.9 mm (score 4), 5-7.9 mm (score 3), 1-4.9 mm (score 2), and not seen (score 1). Metastasis to the bone marrow was assumed in focal lesions with an increased signal in the DWI inverted b-image together with signal loss in the corresponding T1w images (lower than muscle).The standard of reference was either biopsy and histopathology or clinical follow-up. Histopathology was performed in 14 of the 57 patients . Follow-up was used in 43 of the 57 patients supervised by a tumor board consisting of a nuclear medicine physician, a radiologist, and an urologist. A consensus was obtained using information from prior and subsequent imaging studies and/or clinical data . Follow-up consisted of all the clinical information available after study imaging, including clinical examinations, laboratory tests, radiological procedures, and histopathology from surgical procedures performed at a later date. Five patients died from causes unrelated to prostate cancer during the follow-up period. Minimum follow up time per patient was 24 month . Follow-up imaging was done using contrast enhanced CT from thorax-pelvis covering all regions except for the upper and lower extremities. Bone metastases to the extremities were judge in context with other lesions in areas that were covered by CT; there were no solitary or extensive lesions in the extremities that would have required dedicated follow-up imaging. Otherwise judgment of follow-up imaging was based on regions. A significant increase in PSA during follow-up, PSA doubling time < 10 months, confirmation or development of a detectable lesion on follow-up imaging, an increase in lesion size, and a decrease in PSA in response to treatment  were all considered indicative of malignancy [In order to determine the diagnostic value of each method, receiver operating characteristic curves (ROC) were analyzed on a per region basis. The number of regions detected by MRI and 11C-choline PET/CT were compared with the SOR. Cohen’s kappa was used to determine the agreement between the methods on the basis of score points. Agreement was considered to be slight for κ < 0.21, fair for κ = 0.21-0.40, moderate for κ = 0.41-0.60, substantial for κ = 0.61-0.80, and almost perfect for κ = 0.81-1.00 . Sensiti


Selenium and coenzyme Q10 is essential for important cellular functions. A low selenium intake is reported from many European countries, and the endogenous coenzyme Q10 production is decreasing in the body with increasing age. Supplementation with selenium and coenzyme Q10 in elderly have shown reduced cardiovascular mortality and reduced levels of markers of inflammation. However, microRNA analyses could give important information on the mechanisms behind the clinical effects of supplementation.Out of the 443 healthy elderly participants that were given supplementation with 200 μg Se/day as organic selenium yeast tablets, and 200 mg/day of coenzyme Q10 capsules, or placebo for 4 years, 25 participants from each group were randomized and evaluated regarding levels of microRNA. Isolation of RNA from plasma samples and quantitative PCR analysis were performed. Volcano- and principal component analyses (PCA)–plots were used to illustrate the differences in microRNA expression between the intervention, and the placebo groups. Serum selenium concentrations were measured before intervention.-8.On average 145 different microRNAs out of 172 were detected per sample. In the PCA plots two clusters could be identified indicating significant difference in microRNA expression between the two groups. The pre-treatment expression of the microRNAs did not differ between active treatment and the placebo groups. When comparing the post-treatment microRNAs in the active and the placebo groups, 70 microRNAs exhibited significant differences in expression, also after adjustment for multiple measurements. For the 20 microRNAs with the greatest difference in expression the difference was up to more than 4 fold and with a P-value that were less than 4.4eSignificant differences were found in expression of more than 100 different microRNAs with up to 4 fold differences as a result of the intervention of selenium and coenzyme Q10 combined. The changes in microRNA could be a part of mechanisms underlying the clinical effects earlier reported that reduced cardiovascular mortality, gave better cardiac function, and showed less signs of inflammation and oxdative stress following the intervention. However, more research is needed to understand biological mechanisms of the protective effects of selenium and Q10 supplementation. TXNRD) and extracellular selenoprotein P (SEPP 1) are important redox enzymes with a multitude of cellular and extracellular functions such as an anti-oxidative role [Selenium (Se) is essential for a number of cellular functions. In the human selenoproteome 25 separate genes encodes for different selenoproteins. Of thosive role  as well ive role .About half of the selenium in whole blood is found in plasma, of which GPX-3 constitutes 25% and SEPP1 usually constitutes more than 60% . HoweverBesides multiple selenoproteins, coenzyme Q10 is also one of the important antioxidants in the body . The funMicroRNAs, discovered in the beginning of 1990s , 22, proThe aim of the present study was to explore if the intervention with selenium and coenzyme Q10 influenced the expression of circulating microRNAs in the same population.The basal characteristics of the study population is presented in In an epidemiological study in a rural municipality in Sweden that started in 1998, all participants in the age between 70–80 were invited to participate in the intervention study with selenium and coenzyme Q10. Of the 675 in this age span, 443 accepted participation in the study. All participants were examined by one of three experienced cardiologists. In all participants a new clinical history was recorded and a clinical examination was obtained. The New York Heart Association functional class (NYHA class) was also assessed, a Doppler-echocardiographical examination was carried out and an ECG was registered. Blood pressure was measured in the right arm with the participant resting in supine position. All participants were supplemented for 48 months, and were re-examined at the end of each six-month period. All-cause and cardiovascular mortalities were registered.The study protocol has previously been published . The MedThe intervention study was registered at Clinicaltrials.gov, and has the identifier NCT01443780.Diabetes mellitus was defined as having a fasting blood-glucose >7.0 mmol/L, or already undergoing treatment for diabetes . Hypertension was defined as a resting blood pressure more than 140/90 mmHg as measured in the right arm or having a prior diagnosis of hypertension. Dyspnea was defined from the patient history, whereas presence of peripheral edema was defined from patient history and/or clinical examination . IschemiBetween 2003 and 2010 a population consisting of 443 representative elderly individuals were randomized in blocks of 6 in a double-blind manner and given either the active intervention or placebo. Thus 221 persons were given supplement of selenium + coenzyme Q10 , and 200 mg/day of coenzyme Q10 capsules , and 222 persons received the placebo supplement (placebo group) in addition to regular medication. The intervention time was 48 months, and the median follow-up period was 5.2 years.From the total study population of 443 participants, 25 male participants from the active treatment group, and 25 males from the placebo group were randomized and evaluated regarding microRNA, and constitutes the study population of the present study . The micAll blood samples were obtained while the patients were at rest in a supine position. The blood samples were collected in plastic vials containing EDTA (ethylenediamine tetracetic acid). The vials were placed on ice before chilled centrifugation at 3000g and then frozen at -70°C, and no sample was thawed more than twice.Isolation of RNA and real-time quantitative PCR analysis were performed by Exiqon A/S, Vedbaek, Denmark, as previously described .™ RNA isolation kit- biofluids . In brief, 200μL of serum per sample, 60 μL of biofluids lysis solution containing 1μg carrier-RNA and RNA spike-in template mixture was added to the sample. 20μL protein precipitation solution was added and the tube was vortexed, incubated for 1 min at room temperature and centrifuged at 11,000g for 3 min. The clear supernatant was transferred to a new collection tube, and 270μL isopropanolol was added. The solutions were vortexed and transferred to a binding column. The column was incubated for 2 min at room temperature, and emptied using a vacuum-manifold. After that 100μL wash solution was added to the columns, the liquid was removed and 700μL wash solution was added. The liquid was removed using a vacuum-manifold. 250μL wash solution was added and the column was spun at 11,000g to dry the columns completely. The dry columns were transferred to a new collection tube and 50μL RNase free H2O was added directly on the membrane of the spin column to elute bound RNA. The column was incubated for 1 min at room temperature prior to centrifugation at 11,000g. The eluted RNA was stored in a -80°C freezer.Total RNA was extracted from serum using the miRCURY™ UniversalRT microRNA PCR, Polyadenylation and cDNA synthesis kit (Exiqon A/S) [™ Universal RTmicroRNA PCR. Each microRNA was assayed once by qPCR on the microRNA Ready-to-use PCR, serum panel using ExiLENT SYBR® Green master mix. The plasma panel includes 172 microRNAs validated to be significantly expressed in plasma. Negative controls excluding template from the reverse transcription reaction was performed and profiled like the samples. The amplification was performed in a LightCycler® 480 Real-Time PCR System (Roche) in 384 well plates. The amplification curves were analysed using the Roche LC software, both for determination of Cq (by the 2nd derivative method) and for the melting curve analysis.Seven μL RNA was reverse transcribed in 35μL reactions using the miRCURY LNAqon A/S) . cDNA waAll assays were inspected for distinct melting curves and reactions with several melting points are removed from the data set. Reactions with amplification efficiency below 1.6, reactions with Cp values that are within 5 Cp of the negative controls and reactions with Cq>37 are removed from the dataset. microRNA-451 and 23a-3p were analysed to monitor hemolysis.Using NormFinder software, the best normalizer was found to be the average of assays detected in all samples. All data was normalized to the average of assays detected in all samples (average-assay Cq). 145 assays were detected on average per sample. Data analysis was performed according to Exiqon´s recommendation.The serum selenium analyses were performed using ICP-MS methodology at an Agilent 700 platform at Kompetenzzentrum für komplementärmedizinische Diagnostik, Zweigniederlassung der synlab MVZ Leinfelden GmbH . The accuracy of the measurements was checked by analyzing two external reference materials with certified values of 63 μg/L and 103 μg/L , showing values within 90–110% of certified concentrations. A round-robin test with INSTAND e.V. always passed adequately. The precision of the method, checked by repetitive analyses of the same sera, showed an average coefficient of variation (CV) of 5.7%.Descriptive data are presented as percentages or mean and the standard deviation (SD). P-values < 0.05 were considered significant, based on a two-sided evaluation.A Principal Component Analysis (PCA) was used to reduce the dimension of the large data set obtained, and in a PCA plot, the 50 microRNAs that had the largest variation across all samples. The largest component in the variation is plotted along the X-axis, and the second largest is plotted on the Y-axis.Data of microRNA are shown as fold changes between the groups. Two-group comparisons were performed using Student T-test and false discovery rate (FDR) adjustment using the Benjamini-Hochberg (BH) adjusted p-value, correcting for multiple testing. P-values < 0.05 were considered statistically significant.A power calculation has been performed applying 25 participants in each group, and a type I error  of 0.05. Of those 101 mRNAs of the total 145 that obtained significant differences in mRNA expression, all had a power of >0,90, and more than 80% had a power of >0.95 in a two-tailed analysis.All data were analyzed using standard software .The basal characteristics of the two groups, actively treated and placebo groups, for further evaluations of circulating microRNA are presented in A PCR- panel was used for the study, and on average 145 microRNAs were detected per sample.A heat map diagram was produced were the result of a two-way hierarchical clustering of the top 50 microRNAs with the highest standard deviation. The normalized values have been used and the samples are shown . In thisIn the PCA plot , the 50 T-test, 90 microRNAs were found to be differentially expressed using a cut-off P-value <0.05. Of those, 70 were still significant after correction for multiple measurements. T-values of all evaluated mRNAs.Comparing the post-treatment active group to the placebo group using Student´s In There were an increased expression of the microRNA as result of the intervention with selenium and coenzyme Q10 in the following microRNAs; miR-19b-3p, miR-93-5p, miR-16-5p, miR-29b-3p, miR-30e-5p, miR-140-3p, miR-22-3p, miR-363-3p and miR-451a, whereas the following microRNAs showed a decreased expression as a result of the intervention; miR-199a-3p, miR-26a-5p, miR-199a-5p, miR-221-3p, miR-151a-5p, miR-151a-3p, miR-130a-3p,miR-30c-5p, miR-191-5p, and miR-125a-5p.As can be seen, all the 20 microRNAs have a highly significant difference in expression between the post treatment active treatment groups compared to the placebo group.P-values on the y-axis, and the fold change (ddCq) between the two experimental groups on the x-axis so that up- and down regulations appear equidistant from the centre. Plotting the values in this way results in two regions that show microRNAs with the highest magnitude of fold changes and high statistical significance. The highlighted spots are the 20 microRNAs with the greatest difference in expression and with a P-value<0.05 after correction for multiple measurements.In T-test. Only four microRNAs were found to be differentially expressed using a cut-off level of 0.05. However, after applying the Benjamini-Hochberger correction for multiple measurements, none of these differences persisted, indicating that no significant difference existed at the start of the intervention Click here for additional data file.


Our results indicate that dielectric barrier discharge (DBD) plasma, when used with oxygen, can augment the hydrophilicity of non-porous HA surfaces and the osteoconductivity of the IP-CHA disc via increased water penetration of inner porous structures, as demonstrated through microfocus computed tomography (μCT) assay. In vivo implantation of plasma-treated IP-CHA displayed superior bone ingrowth than untreated IP-CHA. Though plasma-treated IP-CHA did not alter osteoblast cell proliferation, it accelerated osteogenic differentiation of seeded marrow mesenchymal stem cells. In vitro X-ray photoelectron spectroscopy (XPS) revealed that this plasma treatment increases levels of oxygen, rather than nitrogen, on the plasma-treated IP-CHA surface. These findings suggest that plasma treatment, an easy and simple processing, can significantly improve the osteoconductive potential of commonly used artificial bones such as IP-CHA. Further optimization of plasma treatment and longer-term follow-up of in vivo application are required toward its clinical application.In the physiochemical sciences, plasma is used to describe an ionized gas. Previous studies have implicated plasma surface treatment in the enhancement of hydrophilicity of implanted musculoskeletal reconstructive materials. Hydroxyapatite (HA) ceramics, widely used in bone tissue regeneration, have made great advancements to skeletal surgery. In the present study, we investigate the impact of low-pressure plasma on the interconnected porous calcium hydroxyapatite (IP-CHA) both  Reconstruction of bone defects within clinical settings, through both autograft and allograft treatments, has achieved varying degrees of success but warrants concerns of donor site morbidity , potentiin vivo rabbit model, the IP-CHA increased in mechanical strength with the formation of new bone, reaching up to three times its initial strength just 9 weeks post-surgery  assay with Cell Counting Kit-8 . MC3T3-E1 cells were cultured in a non-osteogenic standard medium on untreated and plasma-treated HA-coated culture plates (BD BioCoat™ Osteologic™) at an initial density of 3 × 102 for 2 hours. The absorbance was read at 450 nm with an automatic enzyme-linked immunosorbent assay reader .At 1, 2, and 4 day time points, 10 μL of the Cell Counting Kit solution was added into each well, followed by incubation of the culture plates at 37°C in 5% CO2 inhalation, both ends of the femur were removed from the epiphysis; the marrow was flushed out using 10 mL of the aforementioned standard medium expelled from a syringe through a 21-gauge needle according to the method developed by Maniatopoulos et al [2 at 37°C. After 7 days in primary culture, adherent MMCs were released from the culture substratum using 0.1% trypsin. The cells were concentrated by centrifugation at 900 rpm for 5 min at 4°C and resuspended at 106 cells/mL. The untreated and plasma-treated IP-CHA discs were soaked in 4 mL of cell suspension (106 cells/ml) overnight in a CO2 incubator. After the overnight incubation, these MMCs/IP-CHA composites were transferred into a 24-well plate (Corning) for subcultures. DNA content of the MMCs/IP-CHA composite was measured as a cell proliferation assay with IP-CHA discs. Each MMCs/IP-CHA composite was subcultured for 7 days in the non-osteogenic standard medium. After crushing and sonicating each composite, total cellular DNA was extracted using DNeasy Tissue Kit  according to the product manual, and the DNA concentration of the lysate was determined by measurement of the absorbance at 260 nm (A260) in a spectrophotometer .Rat marrow mesenchymal cells (MMCs) were obtained from the bone shaft of the femora of 7-week-old Sprague-Dawley male rats. After sacrifice with COos et al . The relin vitro osteogenic differentiation of MMCs, we subcultured each MMCs/IP-CHA composite in osteogenic medium supplemented with 10 mM of β-glycerophosphate, disodium salt, pentahydrate , 82 μg/ml of L-ascorbic acid phosphate magnesium salt n-hydrate , and 10−8 M of dexamethasone . The medium was renewed three times a week, and the subculture was maintained for 2 weeks. At the end of each subculture period, these MMCs/IP-CHA composites were washed twice with phosphate-buffered saline (PBS), prepared for the staining with alkaline phosphatase (ALP), and evaluated for ALP activity. In the evaluation of the efficacy of in vitro osteogenesis, the subcultures in the non-osteogenic standard medium were referred to as ‘no osteogenic induction’. ALP activity was measured as reported previously [2, and centrifuged at 10,000 rpm for 1 min at 4°C. The supernatant was assayed for ALP activity using p-nitrophenyl phosphate as a substrate. ALP was represented as μmol of p-nitrophenol released per composite for 30 min of incubation at 37°C. The total protein content of those samples, which reflects the cell number, was measured by Bio-Rad Protein Assay  to standardize the ALP activity values.To evaluate how the untreated and plasma-treated IP-CHA discs support eviously , 24. In 2 inhalation. Histology and μCT analysis were performed to evaluate new bone formation in the inner pores.Ten 7-week-old (300–400 g) Sprague-Dawley male rats used in this study were handled in accordance with a protocol approved by the institutional ethical committee. All surgeries were performed under sterile conditions in an animal laboratory surgical suite. Under anesthesia with intraperitoneal injection of a mixture of 0.5 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol, two full-thickness bone defects with a diameter of 5 mm were created symmetrically in the dorsal part of the parietal bone lateral to the sagittal suture using a trephine bur without dura perforation . The plaα line with a photon energy of 1,253.6 eV and an energy spread of 0.7 eV. The pass energy for photoelectrons was set at 75 eV with an photoelectron energy analyzer resolution of 0.1 eV. The pressure in the XPS analysis chamber was set below 2.0 × 10−6 Pa. Prior to each XPS measurement, surface contamination (mostly by carbon) was removed by Ar-beam etching for 2 seconds in the XPS system. Since the diameter of collimated X-ray irradiation region is about 8 mm, the nearly entire area of the IP-CHA surface facing the X-ray source is exposed to the X-ray. Therefore the XPS system provides data averaged over the sample surface. The relative surface concentration of each chemical element was determined from the corresponding high-resolution narrow-scan spectrum, using symmetrical Gaussian peak shapes and integrated background subtraction. The peak positions were determined in the curve fitting process according to tabulated chemical shifts [X-ray photoelectron spectroscopy (XPS) analysis was performed using ESCA-850  in order to determine the chemical composition of IP-CHA after plasma treatment , 27. Thel shifts . XPS meain vitro and the formation of new bone in vivo in each IP-CHA disc, we used μCT system . Each sample was scanned at 10 μm intervals at 50 kV and 200 μA. Analysis was performed in superprecision mode at seven times magnification, with an image intensifier of 1.8 inch in diameter. After scanning, 3D-CT images were reconstructed, and a discoid image  was extracted from the center of each IP-CHA disc. An additional discoid image  of each in vivo sample was extracted from the innermost twelfth region  and the bone formation close to the core of the implant was evaluated. The volume of CM- or bone-filled pores was measured using the TRI3DBON Software package .To evaluate the penetration of CM Decalcified sections were obtained in the following manner; 5 implants harvested from each group at 4 and 8 weeks were fixed in 10% buffered formalin, decalcified in K-CX solution , dehydrated through ethanol series, and embedded in paraffin. 4 μm thick sections were cut parallel to the anatomical coronal planes through the axes of IP-CHA discs, then stained with hematoxylin and eosin (HE) for light microscopy.p = 0.05.Statistical analysis was performed with unpaired t-test with software, STATVIEW version 4.5 . The statistical significance level was set at Water drops placed on non-porous HA surfaces treated with plasma for a duration of 30 minutes spread immediately and their contact angles were too small to be measured. The plasma exposure was therefore reduced to 5 minutes and optimized for contact angle assessment. Photographs of water drops are displayed in The untreated discs remained afloat in water after 10-minute soaking . By contThe WST-8 assay using MC3T3-E1 cells and HA-coating plates demonstrated no significant difference in cell proliferation between the untreated and plasma-treated groups at each time point . IP-CHA After the osteogenic subculture for 14 days, the MMCs/IP-CHA composites were evaluated. ALP staining was more intensively detected in the osteogenic-differentiated group (as seen in ‘untreated’ and ‘plasma-treated’ of 10(PO4)6(OH)2, are also listed as a reference. Quadruplicated (n = 4) XPS measurements were performed for untreated IP-CHA samples and triplicated (n = 3) measurements were performed for plasma treated IP-CHA. It is evident that the surface of an untreated IP-CHA used in this study was not stoichiometric HA. The IP-CHA surface had a higher Ca to P atomic concentration ratio (Ca/P) and a lower O to Ca atomic concentration ratio (O/Ca) than the stoichiometric HA, which suggests that IP-CHA consisted of a mixture of stoichiometric HA and other calcium phosphates [−4 and 8.3 × 10−3 , indicating that the increase of O atoms on the IP-CHA surface by plasma treatment was statistically significant.To examine the change in surface chemical composition of IP-CHA by plasma treatment, we have performed XPS measurements of untreated and plasma-treated IP-CHA sample surfaces. The results are summarized in osphates , 27. It Knowing that the oxygen content on the IP-CHA surface increased significantly by plasma treatment, we now check if there was any other significant change in surface chemical composition by plasma treatment. 43- or OH-). The Ca-O chemical shift seems to have increased after plasma treatment, but it is not conclusive from these findings here. A further study is needed to determine how the increased oxygen is bonded in the plasma treated IP-CHA.in vivo implantation, we performed μCT analysis as previously reported [To measure the total bone volume in the plasma-treated/untreated IP-CHA discs after reported . In Fig 3 vs 4.93 ± 0.57 mm3, P < 0.05). However, the bone volume at 8 weeks did not reach statistical significance . Additionally, between the two groups, the volume of new bone in the innermost twelfth region had more distinctive difference, which reached the statistical significance at 4 weeks but not at 8 weeks , and determined the optimal process parameters for low-pressure, low-frequency plasma processes of IP-CHA . Using taterials .in vivo osteoconductivity of HA-based materials. Although pro-osteogenic modification of HA surfaces was achieved by plasma treatment in vitro, no previous study demonstrated its efficacy in vivo [Plasma treatment can enhance the  in vivo . In the in vivo experiment. Therefore, the enhanced in vivo osteoconduction observed in this study may be due, in part, to the increased water absorbability, along with some pro-osteogenic changes of the material surface [Interestingly, in the core region which comprises the innermost area with a twelfth volume of the whole, the new bone formation was significantly greater at 4 weeks in the plasma group. This trend of superiority was also observed at 8 weeks, despite not reaching statistical significance. The enhancement in osteoconduction by plasma treatment was most noticeable in the core region than in the whole IP-CHA disc. This is due to the interconnected pores of the core which enables sufficient plasma-induced osteoconductivity. Further, considering that the improvement in the innermost region requires the penetration of fluid and cells, the increased water absorbability of IP-CHA discs by plasma treatment may have strongly affected the  surface .-) in a weakly alkaline electrolyte solution [2 plasma. Hence, the difference in findings between our study and the previously published study that implicated nitrogen in the enhanced proliferation of the cells cultured on HA coatings suggests that the efficacy of plasma treatment is highly dependent on the gases used for plasma generation.Changes in the surface chemical composition, which may be responsible for the biological response of cells contacting the material, were elucidated by XPS analysis. The observed increase in concentration of oxygen (O) at the surface of IP-CHA is similar to reports in other studies examining plasma treatment of polymer surfaces , 36, 37.solution . Howeversolution , no subsin vivo osteoconductivity of IP-CHA through increased water absorbability and pro-osteogenic surface modification. The application of plasma to commonly used artificial bones is a simple and easy modification of preexisting biomaterials that can substantially enhance their bone healing and regenerative properties.Our findings suggested that plasma treatment can enhance the in vitro and in vivo performance as a bone substitute. Further optimization of plasma treatment and longer term follow-up of its in vivo application will facilitate its use in a clinical setting.The present study demonstrated that plasma application to IP-CHA, a widely used bone substitute, enhanced its


Introduction. Tuberculosis (TB) is a major disease worldwide, caused by Mycobacterium tuberculosis (MTB) infection. The Toll-Like Receptor (TLR) pathway plays a crucial role in the recognition of MTB. Aim. The present study aimed to investigate the involvement of myeloid differentiation primary response protein 88 (MYD88) gene polymorphisms in TB. Materials and Methods. A total of 103 TB cases and 92 control subjects were genotyped for the MYD88 -938C>A (rs4988453) and 1944C>G (rs4988457) polymorphisms. Results. The MYD88 -938CA and -938AA genotypes were associated with an increased risk for tuberculosis with odds ratio (OR) of 5.71 . Conclusions. The MYD88 -938C>A genetic polymorphism is associated with increased susceptibility to TB and may serve as a marker to screen individuals who are at risk. Mycobacterium tuberculosis, Mtb. TB is responsible for approximately 1.5 million deaths per year , indicating that the risk of disease is related to the mutational load of the variants. In particular, for any two subjects , the probability of being diseased is almost six times higher (relative to the probability of being nondiseased) given that the diseased subject has higher mutational load than the healthy one. Alternatively, a subject has almost six times higher risk of disease relative to the risk of being healthy given that the subject with disease has a higher mutational load than the healthy subject.Subsequently, OR MYD88 -938C>A SNP, the additive and codominant models were tested (p = 0.14) was observed, whereas a significant association (p < 0.01) was shown for the codominant additive model [OR = 5.58 (2.80–11.12)]. We then tested the mode of inheritance for the mutant allele and found that the mutant allele -938A behaves as a dominant allele for the risk of disease (h = 0.79), indicating that the homozygous -938AA has a greater risk of being sick from TB compared to the homozygous -938CC, and that the heterozygous -938CA has a risk of disease closer to the -938AA homozygous than to the midpoint between the two homozygotes (p < 0.05). D primes for LD testing between -938C>A and 1944C>G were 0.80 and 0.59 in cases and controls, respectively.Since significant association was shown for thee tested . A nonsiozygotes . Both SN MYD88 -938C>A, SNP2: MYD88 1944C>G) in patients and control subjects, with the overall difference being significant (p < 0.01). Investigating the individual haplotypes (SNP1-SNP2), A-C, C-C, and C-G also derived significant results (p < 0.01). Haplotype A-C contributes to the risk of disease whereas haplotypes CC and CG may confer protection. MYD88 are associated with TB. According to our results, the MYD88 -938A allele is associated with an approximate 5.5-fold increased risk of TB, whereas no association was found for the MYD88 1944C>G SNP. The genotype distributions of the SNPs examined were all in HW in the control group, indicating no population stratification. Haplotype analysis also showed that the MYD88 -938A- 1944C haplotype conferred an increased risk of disease.The present study investigated whether genetic polymorphisms inIn any case, statistical limitations should be taken into account when interpreting the present findings. The present study constitutes a pilot study, with a small sample size resulting in relatively large ORs. Candidate-gene association studies have the tendency to lack the power to detect a statistically significant association, mainly because of the extremely large population necessary for adequate statistical power. In that context, to achieve a power of 80% to identify a modest genetic effect  of a polymorphism present in 10% of individuals, a sample size of 10,000 subjects or more may be required . This si TLRs genetic variation to TB susceptibility in humans . Severafunction . MYD88 genetic polymorphisms with TB are limited and, in fact, this is the first study investigating the effect of MYD88 -938C>A on susceptibility to TB. Sánchez and colleagues examined the association of MYD88 1944C>G (rs4988457) with TB and, in agreement with our results, found no association with the disease [ MYD88 have been investigated with regard to TB susceptibility. The SNPs rs6767684 and rs7744 were not associated with the disease [Nonetheless, studies examining the association of disease . Various disease , 27, whe MYD88 genetic variation may be associated with susceptibility to TB remains to be elucidated. Data so far show that the MYD88 -938C>A (rs4988453) resides in the promoter region of the gene and has been shown to decrease promoter activity [The exact mechanism through whichactivity . This prMYD88 -938C>A has also been previously associated with sarcoidosis [γ, while reactivation of MyD88 signaling in CD11c or lysozyme M-expressing myeloid cells during Mycobacterium bovis infection is sufficient to restore systemic and local inflammatory cytokine production and to control pathogen burden. TNF-α and IFN-γ are particularly important in promoting the formation and function of the granulomas, whereas IL-10 is one of the main negative regulators of the response in both sarcoidosis and TB [coidosis . Indeed,s and TB , 15, 29. MYD88 -938C>A genetic polymorphism confers susceptibility to TB.In conclusion, our pilot study suggests that the


A progressive deformity associated with adolescent idiopathic scoliosis (AIS) negatively affects a patient’s health-related quality of life (HRQOL). Although the Scoliosis Research Society-22 (SRS-22) is the standard measurement tool for assessing HRQOL in patients with AIS, it is partially suboptimal for evaluating HRQOL in Japanese patients with AIS because of cultural differences. The purpose of this study was to develop a novel patient-reported outcome measure for Japanese female patients with AIS and to evaluate the reliability and validity of this questionnaire in comparison with the SRS-22 tool.We developed 27 questions based on the psychosocial problems in the daily life of young female patients with AIS in Japan, the Scoliosis Japanese Questionnaire-27 (SJ-27). To evaluate its reliability, the internal consistency was assessed using Cronbach’s alpha coefficient. Concurrent validity was evaluated using Spearman’s correlation coefficient between the SJ-27 and the SRS-22. To investigate the construct validity of the SJ-27, the correlation between the SJ-27 questions was assessed using Akaike’s information criterion (AIC).p < 0.001). The AIC analysis indicated that the SJ-27 items are divided into five domains, indicating that the SJ-27 covered a wide range of health-related problems among female patients with AIS.We analyzed 384 female patients with AIS. Cronbach’s alpha coefficients were 0.914 and 0.829 for the SJ-27 and the SRS-22, respectively. Spearman’s correlation coefficient between the SJ-27 and the SRS-22 was 0.692 (The results suggest that the SJ-27 is a reliable and valid patient-reported outcome measure for evaluating HRQOL in female patients with AIS in Japan. A curveThe progressive spinal deformity associated with AIS has been reported to negatively affect a patient’s health-related quality of life (HRQOL) –6, with Patient-reported outcome measures are useful instruments for assessing patients’ HRQOL  and are An original patient-reported outcome measure for Japanese female patients with AIS, the SJ-27, was developed and refined over 17 sessions by an expert committee of the Japanese Scoliosis Society consisting 10 specialists in spinal surgery, rehabilitation, pediatric psychiatrics, and biostatistics. A group of three Japanese Scoliosis Society board-certified experts with extensive spinal disorder treatment experience repeatedly discussed and selected items based on their relevance to the aims of the questionnaire. These experts also sought the advice of a professional pediatric psychiatrist regarding items related to psychosocial problems among adolescent patients with AIS. During the development of the questionnaire, the focus was directed to young female patients because the curve progression and prevalence are greater in females than in males. Additionally, a progressive spinal deformity can result in greater appearance-related psychological problems in females than in males . FurtherFour questions regarding back pain while lying down and after sitting or standing, or neck/shoulder stiffness or soreness (Q1–4)Seven questions regarding discomfort while wearing clothes or holding bags (Q5–10 and 27)Four questions regarding difficulties associated with participating in physical activities Six questions regarding being self-conscious about one’s appearance while in public Six questions related to feelings of anxiety or depression due to the spinal deformity SJ-27 is a self-administered, disease-specific outcome measure that consists of 27 items Table , summariThe SJ-27 is intended to cover a wide range of HRQOL issues among female patients with AIS. The 27 items are scored on a 5-point scale from no impairment (0 point) to severe impairment (4 points) and then added to yield a total score . We adopted the scoring method whereby a higher score indicates a much worse HRQOL, which is opposite to the scoring method of the SRS-22. The reason why we adopted the opposite scoring method to the SRS-22 is because we hope to adopt the same scoring method as the visual analog scale (VAS) or Oswestry disability index (ODI), which are broadly utilized patient-reported outcome instruments in spine disease.Young women 10 to 18 years of age.o.Radiographic findings of a main curve Cobb angle >10Diagnosis of AIS confirmed by experienced specialists.This study is a multicenter cross-sectional analysis designed to assess the reliability and validity of the SJ-27. Female participants with AIS were recruited from outpatient clinics at 24 institutions located throughout Japan between July 2009 and June 2011. These patients were invited to participate in this study regardless of severity and variation in treatment methods. The inclusion criteria were as follows:n = Z2P(1-P)/d2 where n is the sample size, Z is the statistic corresponding to the level of confidence , P is the expected response rate , and d is the precision . Accordingly, the sample size computed using the given formula resulted in 384 participants. We thus aimed to enroll approximately 400 participants in this study.Patients who were unable to respond to the Japanese questionnaire were excluded. The adequate sample size was calculated using the following simple formula : n = Z2Patients invited to participate in this study were given SJ-27 questionnaire booklets and copies of the Japanese version of the SRS-22. It takes approximately 5 min to complete both questionnaires. In the questionnaire instructions, we specified that patients must complete both questionnaires by themselves, but provided that if there were a need for help in filling out the questionnaire, their parents or attendants may help them fill out the questionnaire, but the answer must be patient’s own. The attending physicians recorded the patient’s AIS treatment history, including observation, therapeutic exercise, brace, surgery, and other treatments .Standing anteroposterior radiographs of the whole spine were taken to confirm the AIS diagnosis. The Cobb method was used to measure curve angles at the time the questionnaire was administered.Floor and ceiling effects for the total SJ-27 score and mean total SRS-22 score were examined by calculating the percentage of patients who achieved the lowest  or the highest  score, respectively. We adopted the commonly used 15% threshold for the percentage of patients achieving the lowest or the highest score to define floor and ceiling effects , 42. CroTo evaluate the concurrent validity, the correlation between the SJ-27 and the SRS-22 was assessed using Spearman’s correlation coefficient. The correlation coefficient was interpreted as follows: ±0.1 was considered weak, ±0.3 moderate, and ± 0.5 a strong correlation .http://www.graphviz.org), respectively.To evaluate the construct validity of the SJ-27, the correlation between SJ-27 questions was evaluated using categorical principal component analysis (CATPCA) and Akaike’s information criterion (AIC) , 45. Theo . The AIS treatment history survey showed that 161 patients were treated using braces, 120 were observed, and 78 underwent surgery, respectively.A total of 405 female patients diagnosed with AIS were recruited, and 21 patients over 18 years of age were excluded. Thus, 384 participants were enrolled in this study. Table The floor and ceiling effects for the total SJ-27 score and mean total SRS-22 score were evaluated in this patient group. A total of 370 out of 384 participants (96.4%) completed the SJ-27, while a total of 367 out of 384 participants (95.6%) completed the SRS-22. The mean score in the SJ-27 and the SRS-22 were 23.2  and 4.1 , respectively. The minimum and maximum points of both scales were 0.0 and 86.0 points, respectively, for the SJ-27 and 2.9 and 5.0 points, respectively, for the SRS-22. The percentages of patients who achieved the lowest score were 0.5% for the SJ-27 and 0.0% for the SRS-22, respectively. The percentages of patients who achieved the highest score were 0.0% for the SJ-27 and 0.3% for the SRS-22, respectively.The response completion rates were 99.9% for the SJ-27 and 99.8% for the SRS-22, respectively. Cronbach’s alpha coefficients were 0.914 for the SJ-27 (14 of the 384 participants were excluded due to missing or incomplete answers) and 0.829 for the SRS-22 (17 of the 384 participants were excluded due to missing or incomplete answers), respectively. Table p < 0.001). Four domains were identified using the three-dimensional components plotted with CATPCA  “minimal distance” assortments (degree of independence) for the two-item groupings. Among all the 351 AIC pairs, Q2 and Q3 produced the minimum AIC value (− 252.49), indicating this as the best matched pair. The AIC analysis enabled the division of SJ-27 items into five distinct domains: (1) Pain, (2) Discomfort when wearing clothes, (3) Appearance, (4) Cognition, and (5) Participation, with the five domains being interlinked  completed both the SJ-27 and the SRS-22 questionnaires. Spearman’s correlation coefficient between the SJ-27 and the SRS-22 was 0.692 p < 0.00. Four doA progressive spinal deformity associated with AIS negatively influences a patient’s HRQOL by limiting physical ability and causing back pain, mental disorders, and a negative perception of one’s body –6. A patThe percentage of patients who achieved the lowest score in the SJ-27 and the SRS-22 were 0.5% and 0.0%, respectively. The percentage of patients who achieved the highest score in the SJ-27 and the SRS-22 were 0.0% and 0.3%, respectively. These results indicated that there were no floor and ceiling effects associated with the SJ-27 and the SRS-22 according to the widely used definition of this phenomenon. Based on the results of the current assessment, the SJ-27 showed an excellent internal consistency  and a strong correlation with the SRS-22 . With regard to construct validity, the AIC analysis revealed that five distinct domains  were identified in the SJ-27 , which reflect the typical modest and reserved characteristics associated with Japanese individuals. These characteristics may also be common in other Eastern countries. Furthermore, we excluded the question on economic burden because of the nature of the Japanese medical insurance system. Accordingly, these considerations may lead to the improved reliability and validity of the SJ-27.During the development of the SJ-27, we created seven questions regarding scoliosis-related discomfort when wearing clothes or holding bags (Q5–10 and Q27). These items were independently categorized into a distinct domain (“Discomfort when wearing clothes”) using AIC analysis Fig.  revealedAn analysis of the relationships between items in the SJ-27 using the AIC method revealed that Q17 (“To what extent does your appearance in the mirror make you self-conscious?”) was centered between domains (2) and (4), indicating that Q17 is a key item for assessing HRQOL in female patients with AIS. These patients usually realize and accept their deformity by observing their body shape in a mirror after being diagnosed with scoliosis or when pointed out by family members or colleagues. We speculate that viewing themselves in mirrors may result in self-image problems among adolescent girls and that these constant visible reminders may negatively influence various aspects of the patient’s HRQOL.o) and determine the SJ-27 threshold influencing the decision to initiate therapeutic interventions, especially surgery.The main purpose of using outcome measures is to examine disease clinical severity, determine the proper timing of therapeutic interventions, and choose the most suitable management method for a patient’s condition. The SRS scale has often been utilized for making decisions regarding the initiation of treatment and evaluating the effects of therapeutic interventions. In the future, we wish to evaluate the relationship between the total SJ-27 score and scoliosis severity  has little influence on the internal consistency of the SJ-27 , this instrument may be useful for the assessment of HRQOL in patients with AIS regardless of variation in treatment methods. Second, a healthy control group was not included. Hence, further study is required to validate the SJ-27 by comparing the results of the study group with the results of a control group. Third, some institutions had a small number of female patients with AIS (Table Our study demonstrated that the SJ-27, which is a novel patient-reported outcome measure for Japanese female patients with AIS, showed good reliability and validity. Therefore, the SJ-27 could assist clinicians in assessing their female patients and help individuals with spinal deformities understand their health status.


Polymorphisms, particularly those within the coding regions of proteins and at splice junctions, have received the most attention, but it is also now clear that polymorphisms in the non-coding regions are important. In these non-coding regions, the enhancer and promoter regions have received the most attention, whereas the 3′-UTR regions have until recently been overlooked. In this review, we examine how SNPs affect microRNA-binding sites in these regions, and how mRNA stability changes can lead to disease pathogenesis.In the post-genomic era, the goal of personalized medicine is to determine the correlation between genotype and phenotype. Developing high-throughput genotyping technologies such as genome-wide association studies (GWAS) and the 1000 Genomes Project ( Howeverin silico methods provide novel tools for complex verification of miRNA SNP consequences. In this review, we focus on poly-miRTSs and their potential impact in human diseases.Recently, more attention has been paid to the SNPs identified in non-coding regions. Interestingly, about 93% of functional SNPs in the GWAS catalogue are in non-coding regions . They ha2.2.1.miRNAs are short (approx. 22 nt) endogenous non-coding single-stranded RNAs which act as post-transcriptional regulators of gene expression . In the Although the mechanism underlying the recognition of mRNA targets by miRNAs has been extensively studied, the minimal requirements for a functional mRNA : miRNA association are not fully understood. Furthermore, despite the fact that many mRNAs have conserved miRNA target sites, a variety of interactions through non-conserved sites has been reported . Finallya,b), a 4-mer is a non-functional site in vivo . Be. Be39]. 2.2.2.2.1.MDM4 can lead to a decreased risk of various malignancies [MDM4 has been shown to decrease the risk of cancer, and delay the progression of metastasis and cancer-related death [Mdm2-like p53-binding protein (MDM4) is an oncoprotein that negatively regulates the p53 tumour suppressor protein . It is wgnancies –47. The ed death –47. Numeed death –46 and/oed death ,43,47, aed death .2.2.2.TP63: TAp63 (acidic transactivation domain present) and ΔNp63 (no transactivation domain) [in vivo experiments indicate that TAp63 acts like a tumour suppressor gene, whereas ΔNp63 is an oncogene [et al. [ΔNp63 has an impact on bladder cancer risk. Analysis showed that the T allele is correlated with a decreased risk of bladder cancer because miR-140-5p is able to bind to the 3'-UTR of ΔNp63. Overexpression of miR-140-5p in 5637 cells (urinary bladder grade II carcinoma cells) attenuated cell migration and invasion and inhibited cell proliferation [p63 is another tumour suppressor protein belonging to the p53 family. Because of different promoters and alternative splicing, there are two major isoforms of  domain) . Interesoncogene –75. Wang [et al.  found thferation .2.2.3.(HNF1B) mRNA. This gene encodes a transcription factor known to be a regulator of growth and development in the pancreas [in vivo [et al. [Another example of a positive effect of a SNP on a disease risk is rs2229295 (C>A), which is located in the 3′-UTR of hepatocyte nuclear factor 1-beta pancreas . Since H[in vivo , Goda et [et al.  suggeste [et al. .2.2.4.APOC3 and APOA5 are genes that encode apolipoprotein C3 and A5, respectively. Both of these proteins along with lipoprotein lipase (LPL) and apolipoprotein C2 (APOC2) are involved in triglyceride metabolism [et al. [APOC3. When the T minor allele is present in the cell, miR-4271 is able to bind to the 3′-UTR of APOC3, and this leads to a decreased translation of APOC3. miR-4271, however, cannot bind to the variant containing the G major allele [APOA5 c.*158C>T (rs2266788) is also associated with alterations in triglyceride metabolism and results in hypertriglyceridaemia [APOA5 allele creates a new functional binding site for miR-485-5p. Importantly, both miRNAs regulating APOC3 and APOA5 are endogenously expressed in the human liver, so if the SNP occurs, they may be involved in the regulation of triglyceride metabolism in vivo. However, both examples of SNPs and their impact on the risk of disease need further clarification, since different results have been obtained for different ethnic groups [tabolism ,51. Hu e [et al.  demonstrr allele . Similarc groups .2.2.5.PLIN4 is regulated by miR-522 only in the rs8887A variant. It is not yet clear, however, if the lower expression of PLIN4 contributes to obesity because the results are conflicting [PLIN4 (perilipin 4) is a member of the perilipin family and these proteins coat the intracellular lipid storage droplets (LSD). PLIN4 has been proposed to promote uptake of free fatty acids from the blood to the LSD and is dependent upon the cell's nutritional status . Meta-anflicting ,80.2.2.6.FXN gene [et al. [FXN expression in vivo only in FRDA patients. They identified seven SNPs in the 3′-UTR of FXN in children and adults diagnosed with FRDA. One of them, rs11145043 (G>T), permits miR-124-3p binding only when the T allele is present. Although miR-124-3p is highly expressed in the nervous system [FXN needs further clarification.Reduced expression of the mitochondrial frataxin (FXN) protein has been postulated to play a role in Friedreich's ataxia (FRDA), an inherited neurodegenerative disease . Lower lFXN gene . Additio [et al.  have sugs system , it is os system , suggest2.3.2.3.1.SCNA gene polymorphism is considered a main risk for the common sporadic form of Parkinson's disease  [et al. [The α-synuclein D cases) . α-SynucD cases) . StudiesD cases) . Importa [et al.  have ide [et al. .2.3.2.PALLD gene encodes the actin-associated protein Palladin, whose expression correlates closely with the pathological cell motility characteristics of aggressive cancer cells. The expression level of Palladin in breast cancer patients is higher in invasive and malignant cancer cell types than in non-invasive and normal cell lines. The results suggest that Palladin promotes podosome formation, regulates the actin cytoskeleton via multiple pathways, participates in matrix degradation, and thus facilitates metastasis in breast cancer [et al. [PALLD gene. This SNP is characterized by highest minor allele frequency (greater than 43%) and the alternate G allele is much more common than the ancestral minor C allele. If the C allele occurs in the binding site, the mRNA target sequence at the 3′-UTR of PALLD is fully complementary to the miR-96 and miR-182 seed regions, whereas the presence of the alternate G allele results in one mismatch. A significant decrease in Palladin levels is diminished by miR-96 and miR-182 expression  in the presence of the C allele, but not in the presence of the G allele due to the disrupted miRNA:mRNA association. These findings suggest that although miR-96 and miR-182 may prevent breast cancer metastasis, the functional rs1071738 G variant abolishes this effect [The t cancer ,88. Gila [et al.  have reps effect .2.3.3.EFNB2 (ephrin-B2) gene encodes an ephrin, a protein tyrosine kinase that is involved in remodelling and the development of synaptic connections that are regulated by activated NMDA receptor. Ephrin-B2 is essential for the Reelin pathway controlling neuronal migration. Additionally, the activation of EFNB2 is crucial for rescuing the Reelin defect and disruption of this pathway is associated with schizophrenia [EFNB2 expression was shown [EFNB2. The minor C allele of rs550067317 disrupts the formation of the typical stem-loop structure during base pairing of miR-137 with the predicted target sequence at the 3′-UTR, consequently reversing inhibition of EFNB2 expression.The ophrenia ,89. Receas shown . Importa2.3.4.HIF1A gene encodes the HIF-1α protein (hypoxia-inducible factor 1), an oxygen dependent subunit and master transcriptional regulator of the mammalian cell response to oxygen deprivation, and is therefore important in both the cardiovascular and cancer fields. To date, numerous studies have demonstrated miRNA's role in regulation of HIF-1α levels [HIF1A located near the miR-199a binding site was identified [HIF1A mRNA and consequently upregulation of HIF1 levels, suggesting that this SNP impairs miR-199a : HIF1A binding [The α levels –93. Receentified ,94. The entified . Additio binding .2.3.5.DROSHA gene. The Drosha enzyme, a member of the RNAase III family, plays a critical role in miRNA biogenesis. It liberates the pre-miRNA stem-loop by cleavage of the longer pri-miRNAs in the nucleus [et al. [DROSHA's 3′-UTR contains a target site for miR-27b, while rs10719 (T>C) is located in close proximity to this site (46 bp downstream of the miR-27b binding site). They have postulated that rs10719T to C transition leads to weaker mRNA : miRNA association at the miR-27b target site and consequently to increased Drosha expression.A very interesting example of a synonymous mutation that leads to the loss of an miRNA binding site is SNP rs10719 (T>C) located in the 3′-UTR of the  nucleus . In addi nucleus . Since o nucleus . Yuan et [et al.  reported2.4.2.4.1.et al. [FGF20) and the development of PD. FGF20 is mainly expressed in substantia nigra and increases proliferation and promotes survival of dopaminergic neurons during the early stages of life. However, increased levels of FGF20 in the later stages of life enhance α-synuclein expression and can lead to the death of dopaminergic neurons [FGF20 expression and its relationship to miR-433 levels were confirmed in vivo [An example of another poly-miRTS related to PD was provided by Wang et al. , who rep neurons . miR-433 in vivo .3.The discussed examples of poly-miRTSs strongly suggest that these SNPs can be crucial factors in developing human pathologies and could contribute to genetic diversity. As mentioned, roughly 180 000 SNPs in the human genome that are located in the 3'-UTR region were identified along with about 2600 mature miRNA sequences which are deposited in the mirBase (v. 21), suggesting that a large number of these SNPs may introduce miRNA-binding changes. Furthermore, the recent development of deep sequencing techniques and advanced database/software tools like miRSNP and PolymiRTS Database 3.0 (see in vivo) experimental validation of SNPs related to differential mRNA targeting; (ii) genetic testing of the association with the disease that takes into account the effects of population stratification; and finally (iii) mechanistic testing underlying the mechanism by which poly-miRTSs contribute to the disease [in silico modelling only, ignoring the necessity of the mechanistic approach. Importantly, commonly used methods to confirm differential miRNA : mRNA binding, in vitro luciferase reporter constructs and miRNA overexpression often do not consider the physiological miRNA levels in vivo. However, miRNA physiological levels are often undergoing dynamic changes due to epigenetic factors [in vivo conditions. Hence, in the case of validation of new target sites created by poly-miRTS, this experimental model may lead to false positive results, since it cannot differentiate between weak and strong binding to the targets. The vector-based miRNA expression system that provides inducible and scalable control over miRNA levels may provide more solid verification of potential miRNA : mRNA binding [In 2008, Sethupathy & Collins  provided disease . Few cur factors , and thu binding .Recently, the development of morpholino-based target protector technology provides an elegant tool to test the functionality of novel potential miRNA : mRNA interactions that mimics physiological conditions ,104. TarFurthermore, often changes in a gene's mRNA level are not reflected in its protein levels . Hence, The most convincing and final criterion for linking poly-miRTSs to disease is establishing the disease-related mechanisms of differential miRNA binding. Taking into account complexity of a potential SNP effect on miRNA : mRNA pairing, this can be challenging. Nevertheless, the recent development of targeted genome editing tools (like CRISPR/Cas9 systems) allows one to make efficient, precise and targeted changes to the genome of the living cells, and opens novel possibilities to overcome this limitation . Sadly, in vitro and in vivo disease models could provide the critical proof for the role of and frequency that poly-miRTSs occur in human diseases.Analysing the specific effects of homozygotic and heterozygotic SNPs in both